DESCRIPTION(adapted from applicant's abstract): The problem of protein folding is of great fundamental importance. Two common postulates for how proteins fold efficiently are that conformational preferences-exist in the unfolded state and that folding occurs through intermediates. This proposal focuses on better understanding these non-native states of proteins through careful thermodynamic studies. Specifically, the investigators have developed a methodology that allows mutation-induced changes in the energetics of the denatured state of cytochrome c to be quantified precisely by measuring the thermodynamics of histidine-heme loop formation under denaturing conditions. They have also developed a Lys73-His variant of cytochrome c which stabilizes a partially unfolded state of cytochrome c having properties similar to the lowest energy partially unfolded state of cytochrome c detected by H/D exchange experiments. It is, thus, potentially a model for a late folding intermediate of cytochrome c. The following issues will be addressed with these systems: A set of heme-peptide models and second-site variants of the histidine loop forming cytochromes c will be produced. The effects of local versus long-range interactions and of conformational restriction on denatured state energetics will be addressed with these materials. The dependence of denatured state energetics on denaturant concentration will be assessed to learn how denatured state thermodynamics change when solution conditions favor folding. Experiments on the compactness of the denatured state using Trp59-heme fluorescence energy transfer will be combined with methods which detect equilibrium folding intermediates to assess the role of intermediates versus denatured states in mutationally-induced changes in denaturation m-values. The role of intra- versus inter-substructure interactions in stabilizing the partially unfolded state produced by Lys73-His cytochrome c will be assessed with delta Cp and the m-value for its formation determined by thermodynamic measurements as a function of pH, temperature, and denaturant concentration.